Recently, a high molecular weight (85 kDa) phospholipase A2 (hmw-PLA2) has been purified from mammalian cells and partially characterized. This enzyme may be involved in a signal transduction pathway leading to the generation of free arachidonic acid for eicosanoid biosynthesis. Thus, it may play an important role in a number of inflammatory processes and inhibitors of this enzyme may have therapeutic potential. The enzyme displays specificity toward phospholipids that have arachidonic acid attached to carbon-2 of the glycerol backbone. This specificity will be examined in detail by a competitive protocol in which the enzyme is bound irreversibly to the vesicle surface and is allowed to "choose" between the phospholipids that are present in the bilayer. Phospholipids containing a mixture of different acyl chains of varying length and, double bond number will be synthesized and used in these specificity studies. A second goal is to design a continuous and sensitive assay of the hmw-PLA2. Studies on the design and synthesis of tight-binding inhibitors of the hmw-PLA2 will be carried out and selected compounds will be tested in living cells to see if they modulate the levels of eicosanoid generation. Much of the work will focus on the nature of the phospholipid interface that supports the interfacial binding of the hmw-PLA2. The role of calcium ions in this process will also be examined. Unsaturated fatty acids have been found to enhance the interfacial binding and this phenomenon will be further studied. The amino acid sequence of the hmw- PLA2 contains a region that is homologous to the calcium.dependent phospholipid binding domain of protein kinase C. Interestingly, this latter enzyme is activated by unsaturated fatty acids and this raises the possibility that the activation is due to an enhancement in the interfacial binding. This hypothesis will be tested with purified protein kinase C. All of these studies will be useful in the proper evaluation of activators and inhibitors of the hmw-PLA2.